Use of hepcidin as a regulator of iron homeostasis

ABSTRACT

The invention concerns the use of hepcidin for the diagnosis and therapy of disorders of iron homeostasis. Hepcidin can be used in the treatment of disorders resulting from iron overload while inhibitors of hepcidin can be used in the treatment of anaemia.

This application is a continuation of U.S. application Ser. No.14/747,926, filed Jun. 23, 2015, which is a continuation of U.S.application Ser. No. 11/602,402, filed Nov. 21, 2006, now U.S. Pat. No.9,234,903, which is a divisional of U.S. application Ser. No.10/478,987, filed Jul. 21, 2004, now U.S. Pat. No. 7,169,758, which is aU.S. National Phase Application of International Application No.PCT/EP02/06924, filed May 24, 2002, which claims the benefit of EuropeanApplication No. 01401377.5, filed May 25, 2001, European PatentApplication No. 01401537.4, filed Jun. 14, 2001, and European PatentApplication No. 02290795.0, filed Mar. 29, 2002, all of which are hereinincorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy is named LJH_00703_SL.txt and is 5.96kilobytes in size.

The invention relates to the diagnosis and therapy of disorders of ironhomeostasis.

Iron is an essential element required for growth and survival of almostevery organism. In mammals, the iron balance is primarily regulated atthe level of duodenal absorption of dietary iron. Following absorption,ferric iron is loaded into apo-transferrin in the circulation andtransported to the tissues, including erythroid precursors, where it istaken up by transferrin receptor-mediated endocytosis.Reticuloendothelial macrophages play a major role in the recycling ofiron from the degradation of hemoglobin of senescent erythrocytes, whilehepatocytes contain most of the iron stores of the organism in ferritinpolymers. Over the past five years, an important body of informationconcerning the proteins involved in iron absorption and in theregulation of iron homeostasis has arisen from the study of inheriteddefects, both in humans and mice, leading to distinct iron disorders(for review see ANDREWS, Nat. Rev. Genet., 1, 208-217, 2000). In thecase of iron deficiency, the pathophysiological consequences of genedefects identified are well understood since they usually result in lossof function of proteins directly involved in the pathway of ironabsorption. The proteins include the iron transporters DMT1 (also calledNramp2 or DCT1) (FLEMING et al., Nat. Genet., 16, 383-386, 1997; GUNSHINet al., Nature, 388, 482-488, 1997), ferroportin (also called IREG1 orMTP1) (DONOVAN, et al., Nature, 403, 776-781, 2000), and copper oxidasescoupled to ferroportin, namely ceruloplasmin (HARRIS, Proc. Natl. Acad.Sci. USA, 96, 10812-10817, 1999; YOSHIDA et al., Nat. Genet., 9,267-272, 1995) and haephastin (VULPE et al., Nat. Genet., 21, 195-199,1999).

In contrast, several abnormalities associated with genetic iron overloadhave identified various proteins whose functional role in the control ofiron homeostasis remains poorly understood. In humans, hereditaryhemochromatosis (HH) is a common autosomal recessive genetic diseasecaused by hyperabsorption of dietary iron leading to an iron overload inplasma and multiple organs, including in particular the pancreas, liver,and skin, and resulting in damages in these organs and tissues due tothe iron deposits.

Hemochromatosis is usually due to a mutation in the HLA-linkedhemochromatosis gene (names HFE) located on chromosome 6p, and mostpatients are homozygous for the C282Y mutation in HFE (FEDER et al.,Nat. Genet., 13, 399-408, 1996). In addition, other loci have beeninvolved in different HH families: a nonsense mutation in thetransferrin receptor 2 gene (TFR2) on 7q has been reported in two HHnon-HLA-linked families (CAMASCHELLA et al., Nat. Genet., 25, 14-15,2000) and a locus for juvenile hemochromatosis has recently been mappedto chromosomal arm 1q (HFE2). Finally, although it has long been knownthat iron absorption is regulated in response to the level of body ironstores and to the amount of iron needed for erythropoiesis (ROY et al.,FEBS Lett., 484, 271-274, 2000), this molecular nature of the signalsthat program the intestinal cells to adjust iron absorption stillremains to be identified.

The disruption of the murine gene encoding the transcription factor USF2and its consequences on glucose-dependent gene regulation in the liver(VALLET et. al., J. Biol. Chem., 272, 21944-21949, 1997) have beenrecently reported.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the effects of massive iron overload in the liver andpancreas of Usf2−/− mice. FIG. 1(A) shows a liver section from8-month-old wild-type mice (×50). FIG. 1(B) shows a liver section from8-month-old Usf2−/− littermate. FIG. 1(C) shows a liver section from19-month-old Usf2−/− mouse (×10). The arrowheads in FIG. 1(C) indicateiron in the nucleus of the hepatocyte. FIG. 1(D) shows a pancreassection in from a 8-month-old Usf2−/− mouse (×12, 5). The arrowheads inFIG. 1(D) indicate to islets of Langerhans scattered throughout theexocrine tissue. FIG. 1(E) shows the levels of hepatic iron in mice as afunction of age (in μg iron per gram of dry tissue). FIG. 1(F) shows thelevels of pancreatic iron (in μg iron per gram of dry tissue). In FIGS.1(E) and (F) measurements from wild-type and heterozygote mice areindicated by ▴ and measurements from Usf2−/− mice by □, respectively.

FIG. 2 shows that the spleen of Usf2−/− mice is resistance to naturaliron deposition. FIG. 2(A) shows age-dependent splenic non-heme ironconcentration (micrograms of iron per gram dry tissue) as measured incontrol (wild-type and heterozygote mice, ▴) and Usf2−/− mice (□). FIGS.2(B) and (C) show spleen section from a representative 8-month-oldwild-type mouse (×20) and a 8-month-old Usf2−/− littermate (×20),respectively, stained with the Perls' stain for iron. In FIGS. 2(A) and(B), RP designates red pulp and WP designates white pulp.

FIG. 3 compares the levels of expression of HFE and TFR2 genes in theliver of Usf2−/− mice and wild-type mice. FIG. 3(A) shows a Northernblot measuring the expression levels of HFE and 18S in liver of Usf2−/−mice and wild-type (WT) mice. FIG. 3(B) shows a Northern blot measuringthe expression levels of TfR2 and 18S in liver of Usf2−/− mice andwild-type (WT) mice.

FIG. 4 shows the genomic organization of Usf2 and hepcidin genes. FIG. 4is a schematic representation (not to scale) of the locus regionencompassing the Usf2 and hepcidin genes. The targeted allele isrepresented with the betageo cassette insertion in exon 7. On the rightof FIG. 4 is a Southern blot from tail DNA of wild-type, heterozygoteand homozygote mice.

FIG. 5 shows the level of expression of the hepcidin genes. FIG. 5(A)shows a Northern Blot of total liver RNAs from wild-type, Usf2+/− andUsf2−/− hybridized with a 32P-labeled HEPC transcripts. FIG. 5(B) and(C) show RT-PCR measurement of HEPC1 and HEPC2 levels, respectively.

FIG. 6 shows a proposed model of relating hepcidin to intestinal ironabsorption and macrophage iron stores.

FIG. 7 shows characteristics of TTR-hepc1 transgenic mice. FIG. 7(A)shows a schematic representation of the TTR-hepC1 construction. FIG.7(B) shows a Southern blot with the different founders.

The inventors have now observed that, surprisingly, Usf2−/− mice developmultivisceral iron overload that spares only the spleen whose ironcontent is strikingly lower in knockout animals than in controls. Theseiron metabolic disorders resemble those observed in hereditaryhemochromatosis. However, no alteration in genes previously identifiedfor their implication in this pathology, e.g., HFE or TFR2 was observed.Thus the inventors searched for new candidate genes that may account forthe abnormalities of iron homeostasis in Usf2−/− mice by suppressivesubtractive hybridization between livers from Usf2−/− mice and wild-typemice. This allowed to isolate a cDNA encoding the peptide hepcidin.

Hepcidin (also referred as LEAP-1, for liver-expressed antimicrobialpeptide) was recently purified from human blood ultrafiltrate and fromurine and was found to be a disulfide-bonded peptide exhibitingantimicrobial activity (KRAUSE et al., FEBS Lett., 480, 147-150, 2000;PARK et al., J. Biol. Chem., 276, 7806-7810, 2001). The protein issynthesized in the liver in the form of a propeptide that contains 83amino acids and is converted into mature peptides of 20, 22 or 25 aminoacids (PARK et al., J. Biol. Chem., 276, 7806-7810, 2001; PIGEON et al.,J. Biol. Chem., 276, 7811-7819, 2001). Hepcidin was also recentlyreported to be highly synthesized in livers of experimentally orspontaneously iron overloaded mice (PIGEON et al., J. Biol. Chem., 276,7811-7819, 2001). Although the relationship of this overexpression withiron overload was questioned, it was indicated that it probably resultedfrom inflammation related to chronic iron overload.

In contrast the Inventors have now shown that a complete defect inhepcidin expression leads to a progressive tissue iron overload inUsf2−/− mice. Further they have obtained transgenic mice having atransgene expressing hepcidin under control of a constitutiveliver-specific promoter, and have observed that said transgenic micewere severely anemic.

These findings allow to propose new means of regulation of ironhomeostasis, in particular through regulation of dietary iron capture bythe intestin, or of maternofoetal iron transport through the placentalbarrier, and of iron recycling by reticuloendothelial macrophages.

Accordingly, the present invention proposes the use of a polypeptidecomprising a sequence of 20 amino acids having cysteine residues atpositions 2, 5, 6, 8, 9, 14, 17, and 18, and at least 50% identity or60% similarity, preferably at least 60% identity or at least 70%similarity, with the following sequence:

-   Ile Cys Ile Phe Cys Cys Gly Cys Cys His Arg Ser Lys Cys Gly Met Cys    Cys Lys Thr (SEQ ID NO: 1)

or the use of a nucleic acid encoding said polypeptide, for preparing amedicament useful for reducing iron overload.

Preferred polypeptides or nucleic acids for use according to theinvention are the mature forms of human hepcidin, represented forinstance by a polypeptide of 20 amino-acids having the sequence SEQ IDNO: 1, or by a polypeptide of 22 amino-acids having the sequence;

-   Phe Pro Ile Cys Ile Phe Cys Cys Gly Cys Cys His Arg Ser Lys Cys Gly    Met Cys Cys Lys Thr (SEQ ID NO: 2),

or by a polypeptide of 25 amino-acids having the sequence:

-   Asp Thr His Phe Pro Ile Cys Ile Phe Cys Cys Gly Cys Cys His Arg Ser    Lys Cys Gly Met Cys Cys Lys Thr (SEQ ID NO: 3),

or nucleic acids encoding said polypeptides.

Precursors of said mature forms of hepcidin, i.e. prohepcidin andpreprohepcidin and nucleic acids encoding said precursors can also beused.

Other examples of polypeptides or nucleic acids suitable for useaccording to the invention are vertebrate, preferably mammalian,homologous of mature forms of human hepcidin or precursors thereof, ornucleic acids encoding said polypeptides. Known vertebrate homologous ofhuman hepcidin include for instance rat hepcidin, mouse hepcidin, trouthepcidin.

Chimeric polypeptides, comprising the sequence of a mature form ofhepcidin, (and eventually, all of part of the pro- or theprepro-sequence can also be used.

The invention also encompasses the use of functional equivalents of theabove-defined polypeptides. Functional equivalents are herein defined aspeptide variants, or other compounds having the same functional activityas the mature forms of hepcidin. Examples of such functional equivalentsinclude chemical compounds which are modeled to mimic the threedimensional structure of any of the polypeptides having the sequence SEQID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. Of particular interest arederivatives of said polypeptides having an improved stability andbiological half life. Classical examples of such derivatives are forinstance “retro-inverso” peptides, wherein the sequence of theamino-acids is reversed, and the L-amino acids are replaced with D-aminoacids.

All these polypeptides and nucleic acids can be obtained by classicalmethods known in themselves. For instance, the 20 amino-acids and 25amino-acids forms of hepcidin can be obtained from plasma or from urine,as disclosed by KRAUSE et al. or PARK et al. Alternatively, they can beobtained by culturing cells expressing hepcidin, and recovering saidpolypeptide from the cell culture.

According to a particular embodiment, said cells are host cellstransformed by a nucleic acid encoding one of the polypeptides definedabove.

Chemical synthesis can also be used, in particular in the case of thepeptide derivatives.

A nucleic acid encoding hepcidin can for instance be obtained from agenomic or cDNA library of a vertebrate, using suitable primers able tohybridize selectively with said nucleic acids. It can also be obtainedby the classical techniques of polynucleotide synthesis.

The present invention also provides methods for screening functionalequivalents of hepcidin able to reduce iron absorption.

By way of example, functional equivalents having the biologicalproperties of hepcidin in regulating iron homeostasis can easily bescreened with animals, in particular non-human mammals, lackinghepcidin, for instance knockout mice having a defect in hepcidinexpression resulting in an iron overload.

In particular, a method for screening functional equivalents of hepcidinable to reduce iron absorption comprises the following steps:

-   -   administering to a knockout animal defective in hepcidin        expression a compound to be tested for its ability to reduce        iron absorptions;    -   determining the effect of said compound on iron overload in said        animal.

Medicaments obtained according to the invention are useful forpreventing and/or treating:

-   -   all forms of hemochromatosis;    -   secondary iron overload, related for instance to hereditary        and/or congenital anaemias such as thalassemia;

and diseases associated therewith. These latter diseases include forinstance hepatocarcinoma, cardiomyopathy, or diabetes.

According to another aspect, the invention also proposes the use of aninhibitor of the expression of hepcidin or of the activity of hepcidinfor preparing a medicament useful for increasing iron absorption throughthe increase of dietary iron capture by the intestine, and/or theincrease of iron recycling by the macrophages. Said medicament is usefulfor treating anaemia or anaemia related diseases. This includes inparticular anaemia associated with acute or chronic diseases occurringunder conditions such as infection or inflammation, for instanceosteoarticular diseases such as rheumatoid polyarthritis, ormalignacies, especially when associated with an inflammatory syndrome.

Inhibitors of the expression of hepcidin include for instance antisenseRNA or DNA molecules, or ribozymes.

Inhibitors of the activity of hepcidin include for instanceanti-hepcidin antibodies, in particular antibodies directed against themature forms of hepcidin.

The present invention also provides methods for screening otherinhibitors of the activity of hepcidin, for instance with transgenicanimals, in particular transgenic non-human mammals such as transgenicmice having a transgene expressing hepcidin, said expression inducinganaemia in said animal.

For instance, a method for screening inhibitors of the activity ofhepcidin able to increase iron absorption comprises the following steps:

-   -   administering to a transgenic animal having a transgene        expressing hepcidin a compound to be tested for its ability to        increase iron absorption through the inhibition of the activity        of hepcidin;    -   determining the effect of said compound on anaemia in said        animal.

The medicaments obtained according to the invention can be administeredin various ways, depending on their nature:

For instance, hepcidin polypeptides or functional equivalents thereof,as well as hepcidin inhibitors such as anti-hepcidin antibodies can beadministered by themselves, or mixed with suitable carriers orexcipient(s). They can be used systemically or locally. A preferredroute of administration is the parenteral route, including for instanceintramuscular, subcutaneous, intravenous, or intraperitoneal injections.

The oral route can also be used, provided that the medicament is in aform suitable for oral administration, able to protect the activeprinciple from the gastric and intestinal enzymes.

As indicated above, one can also use a nucleic acid molecule, forinstance a nucleic acid encoding any of the hepcidin polypeptidesmentioned above, in order to enable the expression of said polypeptidein the cells of or a nucleic acid transcribed into an antisense RNA, inorder to suppress the expression of hepcidin in the cells of a subjectto be treated.

In this case, said nucleic acid molecule is introduced into the targetcells by the classical techniques of gene transfer.

Typically, said nucleic acid molecule is placed under transcriptionalcontrol of an appropriate promoter. The choice of the promoter dependson the intended use of the medicament, and/or on the target organ ortissue. Thus one can chose a promoter either constitutive or inductibleand/or either ubiquitous or tissue-specific.

The expression cassette thus obtained can be directly transferred in thecells as naked DNA, or placed in an appropriate vector, such as a viralvector, for instance an adenovirus derived vector.

The choice of the method of transfer and/or of the vector depends on thetarget organ or tissue, and/or on whether a short-time expression(transient expression) or stable expression is wanted.

Gene transfer can be performed ex vivo on cells removed from the subjectto be treated and thereafter re-implanted into said subject, or can beperformed by direct administration of the nucleic acid to said subject.

The invention also provides genetically modified non-human animals,wherein the genetic modification results in an anomaly in hepcidinexpression. The invention also encompasses biological material, such ascells, tissues and organs obtained from said genetically modifiedanimals.

This comprises in particular knockout animals, preferably knockoutmammals, and in particular knockout mice expressing no functionalhepcidin. This lack of expression of hepcidin induces an iron overloadin said animals. The known knockout mices, disclosed by VALLET et al.(J. Biol. Chem., 272, 21944-21949, 1997) wherein the gene encoding thetranscription factor USF2 is inactivated, are excluded.

Knockout animals of the invention are obtainable by total or partialinactivation of the gene(s) of hepcidin, said inactivation resulting inthe absence of production of hepcidin, or in a loss of functionalitythereof.

The inactivation of the gene of hepcidin may target:

-   -   the sequence encoding hepcidin, resulting in the absence of        production of said protein, or in a loss of functionality        thereof, and/or    -   at least one of the regulatory sequences controlling the        expression of hepcidin, resulting in a lack of production of        hepcidin, or in a drastic decrease in the amount of hepcidin        produced.

Other genetically modified animals of the invention having an anomaly inhepcidin expression are transgenic animals, preferably transgenicmammals, and in particular transgenic mice, having a transgeneexpressing hepcidin, said expression resulting in anaemia in saidanimals.

Suitable methods for the preparation of transgenic or knockout animalsare well-known in the art, for instance disclosed in: Manipulating theMouse Embryo, 2^(nd) Ed., by HOGAN et al., Cold Spring Harbor LaboratoryPress, 1994; Transgenic Animal Technology, edited by C. PINKERT,Academic Press Inc., 1994; Gene Targeting: A Practical Approach, editedby A. L. JOYNER, Oxford University Press, 1995; Strategies in TransgenicAnimal Science, edited by G. M. MONASTERSKY and J. M. ROBL, ASM Press,1995; Mouse Genetics: Concepts and Applications, by Lee M. SILVER,Oxford University Press, 1995.

The knockout animals expressing no functional hepcidin, as well astransgenic animals having a transgene expressing hepcidin, can be usedas models for studying the mechanisms of iron homeostasis. They can alsobe used, as described above, for screening of compounds having the sameeffect as hepcidin on iron absorption, or for screening of compoundsable to inhibit the effect of hepcidin on iron absorption.

The invention also provides diagnostic methods for determining whetheran anomaly of iron absorption is associated with a mutation in hepcidinor with an abnormal hepcidin production.

For instance the invention provides:

-   -   a method for detecting whether an anomaly of iron absorption        results from an abnormal hepcidin production, wherein said        method comprises determining the quantity of hepcidin in a        biological sample from a subject suffering from said anomaly;    -   a method for detecting whether an anomaly of iron absorption is        associated with a mutation impairing the production of        functional hepcidin, wherein said method comprises detaching a        mutation in the gene of hepcidin in a nucleic acid sample        obtained from a subject suffering from said anomaly.

Biological samples suitable for determining the quantity of hepcidininclude for instance blood, urine, or amniotic fluid samples, or organbiopsies, in particular liver biopsies or placenta biopsies.

Nucleic acid samples suitable for detecting a mutation impairing theproduction of functional hepcidin include RNA, cDNA or genomic DNA.

The amount of hepcidin in a biological sample can easily be determinedby well-known methods, such as, by way of example, HPLC chromatography,mass spectroscopy, or by immunoassay using anti-hepcidin antibodies.

Mutations in the gene of hepcidin can easily be detected by sequencingsaid gene or a part thereof, previously isolated from the DNA sample tobe tested, and comparing the sequence with the corresponding wild-typesequence(s), obtainable of one or several subjects having no anomaly ofiron homeostasis.

The present invention will be further illustrated by the additionaldescription which follows, which refers to examples illustrating theeffects of the lack of production of hepcidin in knockout animals or ofoverproduction of hepcidin in transgenic animals. It should beunderstood however that these examples are given only by way ofillustration of the invention and do not constitute in any way alimitation thereof.

EXAMPLE 1 Characteristics of Knockout Mice Deficient in HepcidinExpression

Materials and Methods

Generation and Genotyping of Usf2−/− Mice

Disruption of the Usf2 gene has been previously described (VALLET etal., J. Biol. Chem., 272, 21944-21949, 1997). The mutated allelecontains the promoterless IRESβgeo cassette in exon 7 of the murine USF2gene. All studied mice have a mixed genetic background that includedcontributions from C57BL/6 and 129/Sv strains. Mice were maintained on astandard laboratory mouse chow (AO3, UAR, France) containing 280 mg offerric carbonate per kg. Mice were sacrificed from the ages of 2.5months up to 19 months. Genotyping on mouse-tail DNA was performed usinga single PCR reaction in order to identify wild-type (WT) and USF2knockout alleles. Genomic DNA (0.5-1 μg) was used in a 50 μl reactionthat included 3 primers: the wild-type USF2 allele was amplified usingthe following primers:

forward (annealing in intron 6): (SEQ ID NO: 4) GCGAAGCCCTGGGTTCAATC andreverse (annealing in intron 7): (SEQ ID NO: 5) GGGGTCCACCACTTCAAGAGG.

The knockout USF2 allele was amplified using the following primers:

forward: (SEQ ID NO: 6) GCGAAGCCCTGGGTTCAATC, andreverse (annealing in the Neo selection markerof the targeting construct): (SEQ ID NO: 7) GAATTCTCTAGAGCGGCCGGAC.

PCR was performed as follows: 37 cycles (each cycle consisting of 30 sat 94° C., 30 s at 56° C. and 40 s at 72° C.) with an initialdenaturation step at 94° C. for 4 min, and a final elongation step at72° C. for 5 min in 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 0.05% W-I, 2 mMMgCl₂, 5% glycerol, 0.04% bromophenol blue, 0.2 nM each dNTP, 0.2 μMeach primer, 2 units of Taq polymerase (Gibco). The reaction wasanalyzed on 1.5-2% agarose gel containing ethidium bromide. This PCRmethod for mouse genotyping was found to give the same results as theSouthern blot method previously reported (VALLET et al., J. Biol. Chem.,272, 21944-21949, 1997).

Generation of a Subtracted Library by Suppression SubstractiveHybridization (SSH)

Total RNA was prepared as previously described (CHOMCZYNSKI and SACCHI,Anal. Biochem., 162, 156-159, 1987). Polyadenylated RNA was isolatedusing oligo (dT) cellulose (Boehringer Mannheim Biochemica). SSH wasperformed between 3 pooled liver RNA from 5-month-old homozygous USF2deficient mice (‘driver’) and liver RNA from a 5-month-old wild-typemouse (‘tester’) using the PCR-select™ cDNA contraction kit (Clontech)according to the manufacturer's recommendations for all steps. Briefly14 ng of the ligated tester and 420 ng of non-ligated driver cDNAs weremixed, denatured and allowed to re-anneal. After subtractivehybridization, 1 μl of cDNA was amplified by two rounds of PCR. Thesubtracted cDNA library was cloned into the pT-Adv vector using theAdvanTage™ PCR cloning kit (Clontech). After the secondary PCR (15cycles) with the Advantage cDNA polymerase mix (Clontech), thesubtracted PCR cDNA mix was incubated for a further 10 min at 72° C.with 1 unit Tag DNA polymerase (Gibco BRL) to maximize the cloningefficiency and purified with the QIAquick PCR purification kit (Qiagen).The ligation mixture was introduced into the Electromax bacterial strainDH10B (Gibco BRL) by electroporation (1.8 kV) using a Cell-Porator®(Gibco BRL). The library was plated onto 22×22 cm agar plates containingampicillin (100 μg/ml) and spread with 40 μl X-gal (40 mg/ml) and 40 μlIPTG (0.1 M). Bacteria were grown at 37° C. until colonies were visibleand kept at 4° C. until blue/white staining could be clearlydistinguished.

Reverse Northern High Density Blots and Screening

A total of 400 individual clones ware collected, resuspended into 30 μlof water, heated at 100° C. for 10 min, than placed in ice for 5 min andcentrifuged for 5 min. PCR was performed using 3 μl at clear supernatantwith the following primers:

forward: (SEQ ID NO: 8) 5′-CAGGAAACAGCTATGACCATGATTAC-3′, and reverse:(SEQ ID NO: 9) 5′-TAATACGACTCACTATAGGGCGA-3′.

The PCR products were blotted onto Hybond-N+ filters (AmershamPharmacia). Blots were hybridized overnight at 72° C. with¹²P-dCTP-labelled double-stranded cDNA (RTS RadPrime DNA LabelingSystem, Gibco) synthesized with 2 μg polyadenylated from wild-type orUsf2−/− mouse liver, as described below. Blots were washed four times ina 2×SSC/0.1% SDS at 68° C. for 20 min and two times in 0.2×SSC/0.1% SDSat 68° C. for 20 min.

Reverse Transcription and RT-PCR

Double stranded cDNA was synthesized in 20 μl, with 2 μg total RNA (orpolyA RNA for the subtracted library), in the presence of 0.25 mM ofeach dNTP, 200 ng of random hexanucleotide primers, 20 units RNAsin(Promega), 10 mM DTT and 200 units M-MLV reverse transcriptase (Gibco).After denaturation of RNA at 70° C. for 10 min in a therman cycler(Perkin Elmer Cetus), the reaction was performed for 1 hour at 42° C.before reverse transcriptase was inactivated for 6 min at 96° C. At theend of the reaction 80 μl of 10 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA (pH8.0) were added. PCR amplification was performed with 5 μl reversetranscriptase reaction mixture in 50 μl 20 mM Tris-HCl (pH 8.4), 50 mMKCl, 2 mM MgCl₂, 0.05% (v/v) W-1, 0.2 mM of each dNTP, 1 pmol of forwardand reverse specific primers (listed below), 1 pmol of forward andreverse control β-actin primers and 2 units of Taq polymerase (Gibco).PCR conditions were 25 cycles of denaturation at 94° C. for 20 s,annealing at 50° C. for 20 s and primer extension at 72° C. for 20 s.Following PCR, the amplified products (171 bp for HEPC1 and HEPC2 and250 bp for β-actin) were separated by electrophoresis on 1.5% agarosegel.

Sequences of the primers were as follows:

-   *HEPC1:

forward: (SEQ ID NO: 10) 5′-CCTATCTCCATCAACAGATG-3′ and reverse(SEQ ID NO: 11) 5′-AACAGATACCACACTGGGAA-3′; HEPC2: forward:(SEQ ID NO: 12) 5′-CCTATCTCCAGCAACAGATG-3′ and reverse: (SEQ ID NO: 13)5′-AACAGATACCACAGGAGGGT-3′; β-actin: forward: (SEQ ID NO: 14)5′-AGCCATGTACGTAGCCATCC-3′ and reverse: (SEQ ID NO: 15)5′-TTTGATGTCACGCACGATTT-3′.

The primers used for amplification of DMT1 were as follows:

-   *DMT1 isoform without IRE:

forward: (SEQ ID NO: 16) 5′-TCCTGGACTGTGGACGCT-3′ and reverse:(SEQ ID NO: 17) 5′-GGTGTTCAGAAGATAGAGTTCAGG-3′; DMT1 with IRE: forward:(SEQ ID NO: 18) 5′-TGTTTGATTGCATTGGGTCTG-3′ and reverse: (SEQ ID NO: 19)5′-CGCTCAGCAGGACTTTCGAG-3′; Normalization with 14S: forward:(SEQ ID NO: 20) 5′-CAGCACCAAGACCCCTGGA-3′ and reverse: (SEQ ID NO: 21)5′-ATCTTCATCCCAGAGCGA-3′Northern Blot

The primers used for amplification of probes used to detect specificmRNAs were:

* for mouse hemochromatosis (HFE) cDNA amplification (1080 bp):

forward: (SEQ ID NO: 22) 5′-ATGAGCCTATCAGCTGGGCT-3′ and reverse:(SEQ ID NO: 23) 5′-TCACTCACAGTCTGTTAAGA-3′;for mouse transferrin receptor (TfR) cDNA amplification (285 bp):forward: (SEQ ID NO: 24) 5′-GAAATCCCTGTCTGTTATAC-3′ and reverse:(SEQ ID NO: 25) 5′-GGCAAAGCTGAAAGCATTTC-3′;* for mouse transferrin receptor 2 (TFR2) cDNA amplification (333 bp):

forward: (SEQ ID NO: 26) 5′-TACAGCTCGGAGCGGAACG-3′ and reverse:(SEQ ID NO: 27) 5′-TTACAATCTCAGGCACCTCC-3′;for mouse ceruloplasmin cDNA amplification (350 bp): forward(SEQ ID NO: 28) 5′-ACTTATTTCAGTTGACACGG-3′ and reverse (SEQ ID NO: 29)5′-GCAGCACATACACATACTGT-3′; for mouse heme oxygenase 1 (Hmox1) cDNAamplification (258 bp): forward: (SEQ ID NO: 30)5′-ATGGAGCGTCCACAGCCCG-3′ and reverse: (SEQ ID NO: 31)5′-CCTTCGGTGCAGCTCCTCAG-3′.

Each fragment was amplified using Taq polymerase and hepatic total cDNA,purified from agarose gel (QIAquick PCR purification kit, Qiagen) andsubcloned into TA vector (AdvanTAge cloning kit, Clontech). Recombinantplasmid was selected according to the protocol and amplified into LBmedium containing 100 μg/ml ampicillin and purified (QIAprep SpinMiniprep, Qiagen). Each cDNA was purified from the vector after EcoRIdigestion and migration on agarose gel. The probe used to detect HEPC1mRNA was prepared from the EcoRI digestion of the pT-Adv/HEPC1 isolatedby suppressive subtractive hybridization. Twenty micrograms of RNA fromeach source was denatured in formaldehyde-containing buffer andelectrophoreses in 1% agarose, 2.2 M formaldehyde gels. Northern blotwas performed as previously described (VALLET et al., J. Biol. Chem.,272, 21944-21949, 1997). Each blot was stripped and reprobed withribosomal 18 S cDNA, to check for the integrity and the amount of loadedRNAs.

Southern Blot

Southern blots were performed as previously described (VALLET et al., J.Biol. Chem., 272, 21944-21949, 1991). The HEPC1 probe was prepared froma 1437 bp mouse genomic DNA fragment amplified with the followingprimers:

forward: (SEQ ID NO: 32) 5′-GAGCAGCACCACCTATCTCCA-3′ and reverse:(SEQ ID NO: 33) 5′-AACAGATACCACAGGAGGGT-3′.

After digestion with PvuII, a 545 bp fragment was purified from agarosegel and used as probe for Southern blot. This HEPC1 probe showed 95%identity with the homologous HEPC2 region.

Hematological Analysis of Mice

Blood was obtained by retroorbital phlebotomy before sacrifice of miceand collected in heparinized tubes (capiject™ T-MLH, Terumo® medicalcorporation). Blood cell counts and erythrocyte parameters weredetermined using a MaxM coulter automatic analyzer.

Iron Measurement and Histology

Quantification of iron level was performed as previously described byTorrance and Bothwell (1968) on fragments or total organs using IL test™(Instrumentation Laboratory). For histology, tissues were fixed in 4%formaldehyde, embedded in paraffin, mounted onto slides and stained withPrussian blue and nuclear red counterstain using standard procedures.

Results

Massive Iron Overload in Liver and Pancreas of Usf2−/− Mice

All USF2−/− mice exhibit after the third month of life a dense brownpigmentation of the liver and a more or less pronounced bronzepigmentation of the pancreas. As this phenotypic trait is characteristicof hemochromatosis, the inherited disorder of iron absorption, wedecided to analyze the iron status of the Usf2−/− mice. First, to assessthe level of iron accumulation, Perls' Prussian blue staining wasperformed on liver and pancreas of wild-type and Usf2−/− mice maintainedon a standard diet.

The results are shown in FIG. 1(A to D):

Legend: Liver section from (A) 8-month-old wild-type mice (×50), (B)8-month-old Usf2−/− littermate and (C) 19month-old Usf2−/− mouse (×10).Pancreas section in (D) is from a 8-month-old Usf2−/− mouse (×12.5).Arrowheads in C indicate iron in the nucleus of the hepatocyte.Arrowheads in D point to islets of Langerhans scattered throughout theexocrine tissue.

While control mice showed very litter or no positive iron staining inthe liver (FIG. 1A), Usf2−/− mice displayed iron accumulation inhepatocytes (FIG. 1B-C). this iron deposition was primarily confined toperiportal hepatocytes, and then, with age, the number of stainedhepatocytes increased. By 19 months of age, as shown in FIG. 1C, ironaccumulation was considerable and the staining was homogeneousthroughout the liver parenchyma. Furthermore, a strong nuclear ironaccumulation was detected in some hepatocytes (FIG. 1B). For thepancreas, similar results were obtained i.e. no staining in the controltissue and a strong iron accumulation in the exocrine pancreas ofUsf2−/− mice (FIG. 1D).

To quantify more accurately the iron overload during the life ofanimals, iron levels were measured in liver and pancreas of mice from2.5 to 19 months of age.

The results are shown in FIG. 1(E and F):

Legend: Age-dependent hepatic (E) and pancreatic (F) non-heme ironconcentration (micrograms of iron per gram dry tissue) as measured incontrol (wild-type and heterozygote mice, ▴) and Usf2−/− mice (□).

As shown in FIG. 1E, iron accumulated in the liver of mice between 60and 100 days after birth and reached a plateau correspondingapproximately to a 10-fold greater iron content than in wild-type mice.In the pancreas (FIG. 1F), iron accumulation was more progressive, withlevels in Usf2−/− mice a maximum of 20-fold higher than in wild-typemice. Iron accumulation was also measured in kidney and heart showing a2- and 4-fold accumulation, respectively. Finally, a 1.7-fold higheriron level was found in serum of Usf2−/− compared to controlmice(3.550±259 μg of iron/l in controls (n=15) vs 6.274±514 μg of iron/lin Usf2−/− mice (n=13) P<0.0001), but this increase did not appear to beage-dependent. This increase in serum iron level in Usf2−/− mice wascorrelated with a 1.6-fold increase in transferrin saturation (61±9%saturation in controls (n=6) vs 95±9% saturation in Usf2−/− mice (n=6)P<0.0004). Finally, in the oldest female analyzed so far (19 months),the iron overload became widespread with increased iron level in alltissues tested including muscle, uterus, lung and pituitary gland (notshown).

The Spleen of Usf2−/− Mice is Resistant to Natural Iron Deposition

The results are shown in FIG. 2:

Legend of FIG. 2:

(A) Age-dependent splenic non-heme iron concentration (micrograms ofiron per gram dry tissue) as measured in control (wild-type andheterozygote mice, ▴) and Usf2−/− mice (□). Spleen section from arepresentative (B) 8-month-old wild-type mouse (×20) and (C) a8-month-old Usf2−/− littermate (×20 ) stained with the Perls' stain foriron. RP, red pulp; WP white pulp.

In contrast to all other tissues tested, an age-dependent ironaccumulation was observed in the spleen of wild-type mice, as shown(FIG. 2A).

Granules which gave a positive reaction with Perls' Prussian bluestaining were observed, primarily scattered between cells of the redpulp (FIG 2B). We found this accumulation to fluctuate greatly betweenmice, suggesting that it may depend on the (129/Sv×C57BL/6) hybridstrain background of each animal. This natural iron storage has beenpreviously reported in C57BL/6 mice and was described to occur mainly insplenic macrophages (VENINGA et al., Lab. Anim., 23, 16-20, 1989).Surprisingly, in spleen of Usf2−/− mice, iron levels remained very low(FIG. 2A), with a complete absence of Perls' Prussian blue staining(FIG. 2C).

Erythroid Parameters Are Not Affected in Usf2−/− Mice

To rule out the possibility that the increased iron accumulation inUsf2−/− mice might result from dyserythropoietic anemia, erythroidparameters in control and Usf2−/− mice at different ages were measured.Values of red blood cell count (RBC, 10⁶/ml), hemoglobin concentration,(Hb, g/dl) and mean corpuscular volume (MCB, fl) were normal; RBC, Hband MCB of 10.3±0.3, 16.73±0.49 and 48.27±0.67 for wild-type mice (n=3);10.0±0.3, 15.67±0.06 and 48.63±1.36 for Usf2−/− mice (n=3),respectively.

Thus, interestingly, the iron abnormalities observed in Usf2−/− mice,including the resistance of spleen to iron accumulation and normalhematological parameters, strikingly resemble the phenotype of HFE−/−mice (LEVY et al., Blood, 94, 9-11, 1999; ZHOU et al., Proc. Natl. Acad.Sci., USA, 95, 2492-2497, 1998), the murine model of hereditaryhemochromatosis.

Expression of HFE and TFR2 Genes is not Modified in the Liver of Usf2−/−Mice

Because USF2 is transcription factor, it was determined whether USF2could be involved in the regulation of genes encoding proteins relatedto iron metabolism. Due to the similarity between HFE−/− mice andUsf2−/− model, the expression of the HFE gene was first checked. Thegene encoding transferrin receptor-2, a mutation of which was recentlyreported in HH (CAMASCHELLA et al., Nat. Genet., 25, 14-15, 2000) wasalso looked at.

The results are shown in FIG. 3.

Legend of FIG. 3:

Twenty micrograms of total liver RNAs from wild-type mice and Usf2−/−mice (from 3 toll months old) were electrophoresed and blotted. Blotswere hybridized with a 32P-labeled probe (made by PCR, as described inMaterials and Methods) for HFE (A) and Rtf2 (B).

As shown in the Northern blot of FIG. 3A, abundance of HFE mRNA in liverof Usf2−/− mice is comparable to that of wild-type mice. Northern blotanalysis also demonstrated that the hepatic expression of the gene Rtf2was not modified in Usf2−/− mice compared to wild-type mice (FIG. 3B).

The level of ceruloplasmin, heme oxygenase 1 and transferrin receptormRNAs was also monitored in Usf2−/− mice, since the abundance of thesemRNAs has been reported to be modified in disorders that disturb ironbalance (for review see ANDREWS et al., Nutr. Rev., 57, 114-123, 1999).Again it was found that the level of these mRNAs was comparable inUsf2−/− and control mice.

Finally, the expression of the DMT1 gene (also referred to as Nramp2),the major transmembrane iron uptake protein that actively transportsreduced dietary iron into intestinal enterocytes was analyzed. Duodenalexpression of DMT1 was analyzed by relative quantification using RT-PCR(7 Usf2−/− versus 6 control mice). No statistically significantdifferences were found between the two groups of mice (not shown).

Analysis of Subtraction cDNA Libraries: Identification of Hepcidin as aPutative Candidate for Hemochromatosis

To identify genes whose level of expression is modified in Usf2−/− mice,a subtracted cDNA library between liver from Usf2−/− (driver) andwild-type (tester) mice (DIATCHENKO, Proc. Natl. Acad. Sci. USA, 93,6025-6030, 1996) was performed. Among 400 clones analyzed, severalclones were down-regulated in the liver from Usf2−/− mice as analyzed byreverse Northern blot (not shown). One of these clones contained afull-length cDNA encoding the recently characterized peptide hepcidin(KRAUSE et al., FEBS Lett., 480, 147-150, 2000; PARK et al., J. Biol.Chem., 276, 7806-7810, 2001; PIGEON et al., J. Biol. Chem., 276,7811-7819, 2001).

Murine Organization of Usf2 and Hepcidin Genes on Chromosome 7

The murine genome contains two closely related hepcidin genes thatcolocalize on the same mouse genomic clone (Genbank clone, accessionnumber AC020841). These genes were designated HEPC1 and HEPC2 by PIGEONet al. (J. Biol. Chem., 276, 7811-7819, 2001). Interestingly, thegenomic CT7-8N15 clone also revealed that HEPC1 is situated in closeproximity to the Usf2 gene on murine chromosome 7. PIGEON et al reportedthat HEPC1 was located directly downstream of the Usf2 gene (PIGEON etal., J. Biol. Chem., 276, 7811-7819, 2001). By analysing another genomicclone, RP23-22G9 (Genbank, accession number AC087143), it was found thatpart of the Usf2 gene (encompassing exons, 8, 9 and 10) was alsoduplicated and that, in fact, HEPC1 lies downstream of the truncatedUsf2 gene.

The genomic organization of Usf2 and hepcidin genes is shown FIG. 4.

Legend of FIG. 4:

Schematic representation (not to scale) of the locus region encompassingthe Usf2 and hepcidin genes. The targeted allele is represented with thebetageo cassette insertion in exon 7 (VALLET et al., J. Biol. Chem.,272, 21944-21949, 1997). Data are resulting from genomic RP23-22G9 clone(Genbank). So far, no data are available concerning the orientation andthe distance between the two hepcidin genes. The Southern blot in theright of the Figure is from tail DNA of wild-type, heterozygote andhomozygote mice digested by BglII and hybridized with the HEPC1 probe.Two bands of the expected size, 12.4 kbp and 5.1 kbp, were detected,whatever the genotype. The same bands were revealed using the USF2probe.

The HEPC2 gene is located downstream of the functional complete Usf2gene and the HEPC1 gene is located downstream of the partial Usf2 gene.At present, no information is available concerning the relativeorientation, 5′-3′ of the HEPC1 and HEPC2 genes and the distance betweenthem.

Because of the proximity of the Usf2 gene and hepcidin locus, it wasdetermined whether the recombination event in intron 7 of the targetedUsf2 allele might have eliminated or truncated the HEPC1 and HEPC2genes. To check this hypothesis, Southern blot was performed on genomictail DNA from wild-type, Usf2+/− Usf2−/− mice with an HEPC1 probe (FIG.4). Genomic DNA was digested by BglII. Based on the analysis of theAC087143 locus, this digestion was predicted to generate two fragmentsof 5.1 and 12.4 kbp, containing the HEPC1 and HEPC2 genes, respectively.Due to the close similarity (more than 95%) between the hybridizingregion of HEPC1 and HEPC2, both bands were expected to be revealed bythe HEPC1 probe. This is what was found, as shown in the Southern blotin FIG. 4. The same pattern was observed with DNA from Usf2−/− miceindicating that the hepcidin genes are present in Usf2−/− mice and thatthey have not undergone major rearrangement. Finally, the two bands alsohybridized with an USF2 probe extending from exon 8 to exon 10,demonstrating that exons 8 to 10 of USF2 are indeed duplicated.

The Hepcidin Genes are Totally Silent in the Liver of Usf2−/− Mice

The level of expression of the hepcidin genes was measured by Northernblot analysis. In fact, hepcidin mRNA was totally undetectable in theliver of Usf2−/− mice (FIG. 5A). It is worth noting that the liver ofUsf2+/− mice contained a reduced amount of hepcidin mRNA compared withwild-type mice. To further assess the specific level of HEPC1 and HEPC2messengers, specific primers for the HEPC1 and HEPC2 transcripts weredesigned. By RT-PCR it was demonstrated that both genes were activelytranscribed in the liver of wild-type mice (FIG. 5B-C) while both HEPC1and HEPC2 transcripts were totally absent from the liver of Usf2−/− mice(FIG. 5B-C).

Legend of FIG. 5:

(A) Twenty micrograms of total liver RNAs from wild-type, Usf2+/− andUsf2−/− animals (between 3- and 11-month old) were electrophoresed andblotted. The blot was hybridized with a 32P-labeled HEPC probe (preparedas described in “Materials and Methods”) which most likely recognizedboth HEPC1 and HEPC2 transcripts. (B) Specific HEPC1 and HEPC2 levelswere measured by RT-PCR as described in material and Methods. FollowingPCR, the amplified products (171 bp for HEPC1 and HEPC2 and 250 bp forβ-actin) were separated by electrophoresis on 1.5% agarose gel. NeitherHEPC1 nor HEPC2 specific primers were able to reamplify HEPC2 and HEPC1PCR products, respectively, demonstrating the high specificity of eachpair of primers (not shown).

The similarity of the alterations in iron metabolism between HFEknockout mice and the Usf2−/− hepcidin deficient mice suggests thathepcidin may function in the same regulatory pathway as HFE. It has beenshown that HFE physically interacts with the transferrin receptor incrypt cells of the duodenal mucosa (WAHEED et al., Proc. Natl. Acad.Sci. USA, 96, 1579-1584, 1999). Without being bound by theory, it may bepostulated that this interaction modulates the iron status of thesecells which, in turn, controls the expression of the apical andbasolateral transporters in mature cells at the tips of the villi.Hepcidin may be required for HFE activity perhaps through directinteraction with the HFE/beta2 microglobulin/transferrin receptorcomplex. Similarly, hepcidin may be required for the regulation of ironstorage in macrophages. The presence of a mutated HFE protein or acomplete defect in hepcidin expression may be responsible for increasedintestinal iron absorption and reduced macrophage iron stores, accordingto the model shown in FIG. 6.

In this model, hepcidin prevents iron overload by reducing irontransport in the enterocyte and by programming macrophages to retainiron. In Usf2−/− mice, the hepcidin defect would be responsible forincreased intestinal iron transport and reduced macrophage iron stores.

Under both conditions, plasma iron overcomes transferrin bindingcapacity and non-transferrin bound iron accumulates in various tissuesincluding heart and pancreas.

According to the proposed role of hepcidin in iron homeostasis, hepcidinproduction may depend on the uptake of transferrin-bound iron mediatedby TFR2 in hepatocytes. This might explain why the TFR2 defect isresponsible for a form of human genetic hemochromatosis if this defectsleads to a decrease in hepcidin secretion that, in turn, results inincreased iron absorption. This hypothesis will be testable by measuringplasma hepcidin in patients with TFR2 deficiency or in TFR2 knockoutmice.

EXAMPLE 2 Characteristics of Transgenic Mice Overexpressing Hepcidin

Methods

Generation of Transgenic Mice

Full length cDNA of the murine hepc1 cDNA was amplified using primers

(SEQ ID NO: 34) 5′-GGGGGATATCAGGCCTCTGCACAGCAGAACAGAAGG-3′ and(SEQ ID NO: 35) 5′-GGGGGATATCAGGCCTCTATGTTTTGCAACAGATACC-3′.

Both primers contain a StuI site (underlined).

The hepc1 PCR fragment was introduced between the mouse transthyretin(TTR) promoter (consisting of the first exon, first intron and most ofthe second exon) and the SV40 small-T poly(A) signal cassette. Theconstruct carries 3 kb of mouse TTR DNA sequences 5′ to the cap site(YAN et al., EMBO J., 9, 869-879, 1990). The 4.7 kbp TTR-hepc1 transgenewas separated from plasmide sequence by digestion with HindIII and usedfor pronuclear microinjection.

Genotyping by PCR and Southern Blotting

Southern blotting was done according to standard methods (SAMBROOK etal., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press: 1989). Genomic DNA was prepared from tail as follow: a5-mm piece of tail was cut from each mouse and placed into 500 μl ofdigestion mix (50 mM Tris, pH 8/100 mM EDTA/100 mM NaCl/1% SDS).Proteinase K (200 μg) was added and digestion was performed at 55° C.overnight. The samples were extracted directly by adding 500 μl ofphenol/chloroform/isoamyloalcohol (1/24/25). After vortexing andcentrifugation, clear aqueous phase was precipitated with one volume ofisopropanol. For southern blotting, DNA was digested by BamHI, which cuttwice the transgene. After electrophoresis, DNA was transferred to anylon membrane (Hybond-n+, Amersham). Probe corresponds to the 1.7 kbpBglII-HindIII fragment from the previously described TTR plasmid (YAN etal., EMBO J., 9, 869-878, 1990). The probe were labelled with dCTP ³²Pwith random priming, using a commercially available kit (DNA LabelingSystem, Gibco). The 5.3 kbp labelled fragment correspond to endogenousTTR gene and the 4.7 kbp labelled fragment corresponds to the transgene.

For PCR reaction, genomic DNA (0.5-1 μg) was utilized in 25 μl reactionswhich included two primers: TTR-hepc1 transgene was amplified usingprimers:

(SEQ ID NO: 36; annealing in intron 1 of TTR)5′-CTTTTTGCACCATGCACCTTTC-3′ and(SEQ ID NO: 37; annealing in hepc1 cDNA) 5′-AACAGATACCACACTGGGAA-3′.

PCR reaction was performed as following: 25 cycles (each cycle consistof 40 seconds at 94° C., 40 seconds at 50° C. and 40 seconds at 72° C.)with an initial denaturation step at 94° C. for 4 minutes and a finalelongation step at 72° C. for 5 minutes in 20 mM Tris-HCl (pH 8.4), 50mM KCl, 0.05% W-1, 2 mM MgCl₂, 0.2 mM each dNTP, 0.2 μM each primer, 2units of Taq polymerase (Gibco). The 612 bp specific product wasamplified with a non specific fragment of the same size. The presence ofthe transgene is revealed after digestion of the PCR product with 10units of StuI during 2 hours that produces 268 bp and 344 bp. Reactionwas analyzed on 1.5-2% agarose gel containing ethidium bromide. Theamplification of the non specific fragment ensure that the absence oftransgene is not due to the lack or degradation of genomic DNA. This PCRmethod for mouse genotyping was found to give the same results as theSouthern blot method.

Results

Characteristics of TTR-hepc1 Transgenic Mice

A total of nine independent transgenic founder mice were produced byclassical microinjection method of a linearized construct.

The construct is schematically represented in FIG. 7A.

FIG. 7B shows a Southern blot with the different founders.

Three transgenic mice founders (TH27, TH37, and TH52) wereindistinguishable from their wild-type mice (Wt) littermate. Threetransgenic mice founders were born with a pallor skin and died within afew hours after birth (bb2, 3 and 5). Finally, the phenotype of thethree last transgenic mice founders (TH5, 35, and 44) was unambigous;they had a hairloss on whole body and their skin was crumpled. Bloodsmears were performed on these animals and evidences of strongpoikylocytosis and hypochromia were found in the three mice with thecrumpled skin.

The above examples highlight the role of hepcidin as a key regulator ofiron homeostasis. Hepcidin is proposed as a novel candidate gene that,when mutated, could be involved in abnormal regulation of ironmetabolism and development of HH. Finally, the new murine model of ironoverload disease disclosed above appears to be a suitable animal modelfor testing new therapeutic approaches for prevention and correction ofthe iron storage in HH as well as for the understanding of ironhomeostasis.

The invention claimed is:
 1. A method for treating hereditaryhemochromatosis in a patient in need thereof, comprising administeringto the patient a polypeptide comprising a sequence of SEQ ID NO:
 1. 2. Amethod according to claim 1, wherein the polypeptide comprises the aminoacid sequence set forth in SEQ ID NO:
 2. 3. A method according to claim1, wherein the polypeptide comprises the amino acid sequence set forthin SEQ ID NO:
 3. 4. A method according to claim 1, wherein thepolypeptide consists of the amino acid sequence set forth in SEQ ID NO:3.
 5. A method according to claim 1, wherein the polypeptide isadministered parenterally.
 6. A method according to claim 5, wherein thepolypeptide is administered by intramuscular, subcutaneous, intravenous,or intraperitoneal injection.
 7. A method according to claim 4, whereinthe polypeptide is administered parenterally.
 8. A method according toclaim 7, wherein the polypeptide is administered by intramuscular,subcutaneous, intravenous, or intraperitoneal injection.
 9. A method fortreating anemia in a patient in need thereof, comprising administeringto the patient a polypeptide comprising a sequence of SEQ ID NO:
 1. 10.A method according to claim 9, wherein the polypeptide comprises theamino acid sequence set forth in SEQ ID NO:
 2. 11. A method according toclaim 9, wherein the polypeptide comprises the amino acid sequence setforth in SEQ ID NO:
 3. 12. A method according to claim 9, wherein thepolypeptide consists of the amino acid sequence set forth in SEQ ID NO:3.
 13. A method according to claim 9, wherein the polypeptide isadministered parenterally.
 14. A method according to claim 13, whereinthe polypeptide is administered by intramuscular, subcutaneous,intravenous, or intraperitoneal injection.
 15. A method according toclaim 12, wherein the polypeptide is administered parenterally.
 16. Amethod according to claim 15, wherein the polypeptide is administered byintramuscular, subcutaneous, intravenous, or intraperitoneal injection.17. A method for treating thalassemia in a patient in need thereof,comprising administering to the patient a polypeptide comprising asequence of SEQ ID NO:
 2. 18. A method according to claim 17, whereinthe polypeptide comprises the amino acid sequence set forth in SEQ IDNO:
 3. 19. A method according to claim 17, wherein the polypeptideconsists of the amino acid sequence set forth in SEQ ID NO:
 3. 20. Amethod according to claim 17, wherein the polypeptide is administeredparenterally.
 21. A method according to claim 20, wherein thepolypeptide is administered by intramuscular, subcutaneous, intravenous,or intraperitoneal injection.
 22. A method according to claim 19,wherein the polypeptide is administered parenterally.
 23. A methodaccording to claim 22, wherein the polypeptide is administered byintramuscular, subcutaneous, intravenous, or intraperitoneal injection.